EP2005161B1 - Capteur electrochimique - Google Patents

Capteur electrochimique Download PDF

Info

Publication number
EP2005161B1
EP2005161B1 EP07718585.8A EP07718585A EP2005161B1 EP 2005161 B1 EP2005161 B1 EP 2005161B1 EP 07718585 A EP07718585 A EP 07718585A EP 2005161 B1 EP2005161 B1 EP 2005161B1
Authority
EP
European Patent Office
Prior art keywords
binding moiety
redox active
active species
sensor
electrode
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Not-in-force
Application number
EP07718585.8A
Other languages
German (de)
English (en)
Other versions
EP2005161A1 (fr
EP2005161A4 (fr
Inventor
John Justin Gooding
Guozhen Liu
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
NewSouth Innovations Pty Ltd
Original Assignee
NewSouth Innovations Pty Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from AU2006901394A external-priority patent/AU2006901394A0/en
Application filed by NewSouth Innovations Pty Ltd filed Critical NewSouth Innovations Pty Ltd
Publication of EP2005161A1 publication Critical patent/EP2005161A1/fr
Publication of EP2005161A4 publication Critical patent/EP2005161A4/fr
Application granted granted Critical
Publication of EP2005161B1 publication Critical patent/EP2005161B1/fr
Not-in-force legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
    • G01N33/5438—Electrodes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/817—Enzyme or microbe electrode

Definitions

  • the present invention relates to electrochemical sensors for detecting the presence of an analyte in a sample.
  • a number of electrochemical techniques for detecting the presence of an analyte in a sample have been described. These techniques can be classified as catalytic, where a reaction of a modified electrode with the analyte produces a new species which can be detected electrochemically, or affinity based, where a binding reaction between the analyte and a binding partner is detected electrochemically.
  • redox-labelled species that enables differentiation between before and after binding of the analyte.
  • the redox-labelled species, labelled analyte and/or other reagents are added to the sample at some stage during the analysis in order for the binding event to be detected electrochemically. It is therefore necessary for the person using the sensor to intervene during the analysis. The need for user intervention necessitates that such sensors require skill to operate.
  • EP 0 402 126 A discloses an electroactive complex characterised in that it comprises at least one antigenic analyte chemically-bonded to a carrier molecule, the carrier molecule also being chemically-bonded to at least one electroactive molecule.
  • KWON et al "An electrochemical immunosensor using ferrocenyl-tethered dendrimer"; Analyst; viol.131, 2006, pages 402-406 , discloses an enzyme-amplified sandwich-type immunosensor for detecting the biospecific interaction between an antibody and antigen using redox mediation, employing a biotin/anti-biotin IgG as a model immunosensing pair.
  • WO 00/11474 discloses an electrochemical affinity assay system for detection of ligand-ligand receptor binding.
  • US 5,534,132 discloses an amperometric biosensor for the detection of an affinity reaction, the sensor including an electrode having on its testing surface a hydrogel in which a selective binding unit and redox species are immobolised.
  • the present invention provides an electrochemical sensor for detecting an analyte, the sensor comprising:
  • the binding moiety when the binding moiety associates with the analyte, the association of the binding moiety and analyte remains in situ, thereby affecting the electrochemistry of the redox active species.
  • the binding moiety when the binding moiety associates with the analyte, the binding moiety is released from the sensor, and the release of the binding moiety affects the electrochemistry of the redox active species.
  • the electrochemical sensor of the present invention exploits the changes in electrochemistry of the redox active species which occur when the binding moiety associates with the analyte in order to detect the presence of the analyte in a sample.
  • the redox active species is electrochemically accessible to the electrode, it is provided as part of the sensor and the changes in its electrochemistry occur without the need to add additional redox active species (or species capable of reacting to generate a redox active species) during analysis of a sample.
  • the changes in the electrochemistry of the redox active species can be detected electrochemically contemporaneously with the association of the binding moiety with the analyte.
  • the binding moiety is bound to the redox active species.
  • the present invention provides an electrode, wherein a redox active species is bound to the surface of the electrode by a species that is a conduit for electron movement, and a binding moiety capable of associating with an analyte is associated with the redox active species, the binding moiety being sufficiently proximate to the redox active species such that association of the binding moiety with the analyte affects the electrochemistry of the redox active species, and wherein association of the binding moiety with the analyte affects the electrochemistry of the redox active species and is detectable by amperometric electrochemical measurements, wherein a change in the electrochemistry of the redox active species occurs when the binding moiety associates with the analyte.
  • the binding moiety may be associated with the redox active species in any manner such that association of the binding moiety with the analyte affects the electrochemistry of the redox active species. Typically, the binding moiety is bound to the redox active species.
  • the electrochemical sensor is for detecting an antibody
  • the sensor comprises:
  • the binding moiety is preferably at least part of an antigen (i.e. an antigen to which the antibody is capable of binding).
  • the binding moiety comprises an epitope capable of becoming bound to the antibody.
  • the binding moiety may consist of the epitope.
  • the binding moiety may be the antigen itself.
  • the binding moiety typically has a very high degree of selectivity for the antibody, and the sensor is therefore less susceptible to interference from other species which may be present in the sample tested.
  • the epitope may be chemically synthesized. That is, the binding moiety may be a chemical analogue of an epitope of an antigen.
  • the electrochemical sensor is for detecting an antigen
  • the sensor comprises:
  • the second sensor further comprises at least part of an antigen (i.e. an antigen to which the antibody is capable of binding), and the binding moiety is bound to the at least part of an antigen.
  • an antigen i.e. an antigen to which the antibody is capable of binding
  • the binding moiety is bound to the at least part of an antigen.
  • the at least part of an antigen is bound to the redox active species and to the antibody binding moiety.
  • the at least part of an antigen may comprise an epitope of the antigen.
  • the at least part of an antigen is typically bound to the redox active species (either directly or, for example, by a short length alkyl chain).
  • the at least part of an antigen is bound to the redox active species, and the binding moiety is bound to the at least part of an antigen.
  • the electrode when the electrode is placed in a sample containing the antigen, at least some of the antibody binding moiety binds to the antigen in the sample and is released from the at least part of an antigen (i.e. released from the sensor).
  • Such sensors typically have a high degree of selectivity for the antigen, and have little susceptibility to interference from other species which may be present in the sample.
  • the antibody When the first sensor is exposed to a sample that contains the antibody, the antibody binds to the binding moiety.
  • the inventors believe that the electrochemistry of the redox active species is suppressed following binding, because ions in the sample are restricted from interacting with the redox active species as the relatively large antibody enfolds the redox active species and effectively "shields" the redox active species from ions in the sample. This suppression is detectable using electrochemical techniques and may be quantified.
  • transduction of the affinity based recognition event i.e. the antibody attaching to the binding moiety
  • the second sensor When the second sensor is exposed to a sample that contains the antigen, a competition for the binding moiety (i.e. the antibody) occurs between the sensor and the antigen in the sample. As a result of this competition, at least some of the antibody binding moieties detach from the sensor, which results in the redox active species becoming more exposed to ions in the sample. Again, this is detectable using electrochemical techniques, and transduction of the affinity based recognition event may be achieved without user intervention.
  • the binding moiety i.e. the antibody
  • the redox active species is bound to the electrode by a species that is a conduit for electron movement, for example, a molecular wire or a nanotube, both of which are rigid species that efficiently transfer electrons.
  • the electrochemical sensor of the present disclosure typically further comprises blocking agents, for example polyethylene glycol (PEG), bound to the surface of the electrode.
  • blocking agents for example polyethylene glycol (PEG) bound to the surface of the electrode.
  • PEG polyethylene glycol
  • the electrochemical sensor of the present disclosure typically further comprises a detector capable of detecting changes in the electrochemistry of the redox active species as a result of association of the binding moiety with the analyte.
  • the change in the electrochemistry of the redox active species is typically detected by analysing changes in the ability of the electrode to oxidise and reduce the redox active species as the potential of the electrode is scanned anodically and cathodically respectively.
  • the electrochemical sensor of the present invention typically further comprises an electrical power source and a display for displaying electrochemical readings from the electrode.
  • the present invention provides a method for detecting the presence of an analyte in a sample, the method comprising the steps of:
  • the method is for detecting the presence of an antibody in a sample, the method comprising the steps of exposing to the sample a binding moiety capable of becoming bound to the antibody, the binding moiety being associated with a redox active species that is electrochemically accessible to an electrode; and taking electrochemical measurements which indicate whether there is a change in the electrochemistry of the redox active species as a result of the antibody binding to the binding moiety.
  • the method is for detecting the presence of an antigen in a sample, the method comprising the steps of exposing to the sample a binding moiety that is an antibody capable of binding to the antigen, the binding moiety being associated with a redox active species that is electrochemically accessible to an electrode; and taking electrochemical measurements which indicate whether there is a change in the electrochemistry of the redox active species as a result of the binding moiety disassociating from the redox active species.
  • the method of the first embodiment of the third aspect of the present invention may be carried out using the first sensor.
  • the method of the second embodiment of the third aspect of the present invention may be carried out using the second sensor.
  • a method for detecting the presence of an analyte in a sample comprising the steps of exposing to the sample the electrochemical sensor of the first aspect of the present invention; and taking electrochemical measurements which indicate whether there is a change in the electrochemistry of the redox active species as a result of association of the binding moiety with the analyte.
  • the electrochemical measurements which indicate whether there is a change in the electrochemistry of the redox active species may be made during or after association of the binding moiety with any of the analyte present in the sample.
  • the method consists essentially of, or consists only of, the steps referred to above.
  • the electrochemical sensor of the first embodiment of the first aspect of the present invention is for detecting an antibody.
  • the first sensor comprises an electrode, a redox active species that is bound to the electrode by a species that is a conduit for electron movement, and is electrochemically accessible to the electrode, and a binding moiety capable of becoming bound to the antibody.
  • the sensor when the sensor is exposed to a sample which contains the antibody, the antibody binds to the binding moiety and the electrochemistry of the redox active species is affected.
  • a reference to exposing a sensor of the present invention to a sample refers to exposing the sensor to the sample in a manner that would permit the binding moiety to associate with any of the analyte that may be present in the sample.
  • the binding moiety is bound to the redox active species, and the sensor is exposed to the sample by placing at least part of the electrode in the sample, thereby enabling the binding moiety to associate with any of the analyte present in the sample.
  • the electrochemical sensor of the second embodiment of the first aspect of the present invention is for detecting an antigen.
  • the second sensor comprises an electrode, a redox active species that is bound to the electrode by a species that is a conduit for electron movement, and is electrochemically accessible to the electrode, and a binding moiety that is an antibody capable of binding to the antigen.
  • a redox active species that is bound to the electrode by a species that is a conduit for electron movement, and is electrochemically accessible to the electrode
  • a binding moiety that is an antibody capable of binding to the antigen.
  • the second sensor further comprises at least part of an antigen to which the antibody binding moiety is capable of binding (an "antigen analogue"), and the binding moiety is bound to the antigen analogue on the sensor.
  • an antigen analogue an antigen to which the antibody binding moiety is capable of binding
  • the second sensor may be essentially the same as embodiments of the first sensor in which the binding moiety is an antigen analogue, except that an antibody is bound to the antigen analogue.
  • the specific embodiments of the second sensor described below will include an antigen analogue.
  • the senor When using the second sensor, if the antigen is present in the sample, the sensor will show an increase in electrochemistry. Thus, a positive signal occurs when the electrode of the second sensor is exposed to a sample containing the antigen and an electrical current is passed through the electrode. In general, sensors which provide a positive signal are more desirable because they are less prone to giving false positive results.
  • the present invention can provide a user-intervention-free immunobiosensor which exploits the modulation in the electrochemistry of a redox active species provided as part of the sensor by antibody binding or detachment to/from the sensor.
  • Schematic representations of the surface of an electrode for embodiments of the first and second sensors (labelled “First Sensor” and “Second Sensor” respectively) are shown in Figure 1 .
  • Illustrative cyclic voltammograms for each sensor are depicted beside the respective representation.
  • the redox active species is labelled "redox probe”.
  • the cyclic voltammograms show the attenuation of the electrochemistry of the redox probe upon the antibody binding to the sensor.
  • the antibody in these representations is depicted for clarity as being approximately only slightly larger than the antigen. The antibody would typically be many times larger than the antigen.
  • the sensors of the present invention will typically include a large number of redox active species and binding moieties distributed on the surface of the electrode in order for the association of the binding moiety and the analyte to cause a detectable change in the electrochemistry of the redox active species.
  • Electrodes suitable for use in the sensors of the present invention include, for example, carbon paste electrodes, screen-printed carbon electrodes, glassy carbon (GC) electrodes, gold electrodes, platinum electrodes, carbon nanotube electrodes, indium tin oxide electrodes, silicon electrodes, aluminium electrodes, copper electrodes, etc.
  • GC electrodes are used because they are inexpensive and can be mass produced. They are also very dense, chemically inert, electrically conductive and have a relatively well defined structure. GC electrodes can also be modified by the formation stable self assembled monolayers (SAMs) on the surface of the electrode using techniques described in the art. Modified GC electrodes have a large potential window, which is advantageous because it allows many different types of molecules to be investigated electrochemically (some molecules are not stable at too negative or too positive potentials).
  • SAMs stable self assembled monolayers
  • the redox active species may be any species that can be electrochemically interrogated.
  • the redox active species must be electrochemically accessible to the electrode in order for electrons to be transferred between the species and the electrode, so that changes in the redox state of the redox active species can be detected by changes in the electrical current through the electrode.
  • the binding moiety e.g. antigen
  • the binding moiety may itself contain a redox active species.
  • the sensor need not have an additional redox active centre (i.e. the redox active species is part of the binding moiety).
  • the at least part of an antigen may itself contain a redox active species and the sensor need not have an additional redox active centre.
  • Suitable redox active species include organometallic complexes, metal ion complexes, organic redox active molecules, metal ions and nanoparticles containing a redox active centre.
  • the redox active species is chemically bound to the distal end of a species that is a conduit for electron movement (as will be described below), and therefore bound, via the species that is a conduit for electron movement, to the electrode.
  • the redox active species and the binding moiety are situated sufficiently proximate to each other so that the association of the binding moiety with the analyte affects the electrochemistry of the species.
  • the redox active species may be directly bound to the binding moiety.
  • the redox active species may be bound to the binding moiety via a short (e.g. C 1-10 ) alkyl chain, or the like.
  • the redox active species is bound to the binding moiety via a C 1-5 alkyl chain.
  • the redox active species in the sensor of the present invention is typically the redox active centre in a redox active compound, where the redox active compound is capable of undergoing chemical reactions in order to bind the redox active centre to other components of the sensor.
  • Compounds that may be used typically have one or more functional groups that enable them to bind to other components of the sensor (e.g. the binding moiety or species that is a conduit for electron movement) via chemical bonds.
  • Preferred redox active compounds that may be used possess amine functional groups, which facilitate the attachment of the compound to other components of the sensor.
  • ferrocenedimethylamine and flavin adenine dinucleotide are redox active compounds that can react with, and be covalently attached to, other compounds via amide coupling(s).
  • the redox active centre in ferrocenedimethylamine is referred to below as the "ferrocene moiety”.
  • Specific examples of compounds that may be used to incorporate the redox active species in the sensor of the present invention include ferrocenedimethylamine, 1,5-diaminonaphthalene, pyrrolo quinoline quinone, 2,3,5,6-tetramethyl-1,4-phenylenediamine, flavin adenine dinucleotide, ethidium, ruthenium(NH 3 ) 4 pyridine 2+ , ruthenium(2,2'bipyridyl) 2 (dipyrido[3,2: ⁇ -2',3': ⁇ ]phenazine) 2+ , ruthenium((5-glutaric acid monohydride)-1,10-phenanthroline) 2 (dipyrido[3,2: ⁇ -2',3': ⁇ ]phenazine) 2+ , ruthenium(2,2'-bipyridyl) 4 (imidazole)(2-amino-2-deoxyuridine), rhodium(9,10-phen
  • the redox active species must be electrochemically accessible to the electrode in order for changes in the redox state of the species to be detected by changes in the electrical current in the electrode.
  • the redox active species is bound to the electrode via a species that is a conduit for electron movement.
  • the species that is a conduit for electron movement provides a means by which electrons can move between the redox active species and the electrode, for example by tunneling or electron transport.
  • Examples of species that can be conduits for electron movement include molecular wires, nanotubes (such as single walled carbon nanotubes) and norbornylogous bridges.
  • Molecules that may be used to form a conduit for electron movement between the electrode and the redox active species include aliphatic alkanes, oligo(phenylene vinylene), oligo(phenylene ethynylene), polyacetylene, polythiophene, Carotenoids and Li 2 Mo 6 Se 6 .
  • the species that is a conduit for electron movement is bonded to the surface of the electrode at one of its ends.
  • the species is substantially linear, and its other end is bonded to the redox active species.
  • the ends of a compound that can be used to create the species that is a conduit for electron movement in a sensor of the present invention preferably have functional groups (e.g. carboxylic acid functional groups) that are capable of reacting with another compound.
  • a redox active species possessing an appropriate functional group e.g. an amine functional group
  • a second reaction such that the electrode and redox active species are joined by the species that is a conduit for electron movement.
  • a combination of one or more different types of species that are conduits for electron movement may be used in the sensor.
  • the binding moiety in the electrochemical sensors of the present invention is capable of associating with the analyte, which results in the electrochemistry of the redox active species being affected.
  • the association of the binding moiety and analyte is affinity based, that is, the binding moiety and analyte have an affinity for binding to each other.
  • the binding typically occurs as a result of the binding moiety or the analyte (the "recognition molecule”) having the correct geometric shape for the analyte or the binding moiety to bind whereupon a combination of weak bonding forces, such as hydrogen bonding, van der Waals forces and electrostatic forces, operate cooperatively to strongly bind the analyte and binding moiety together.
  • weak bonding forces such as hydrogen bonding, van der Waals forces and electrostatic forces
  • the sensors of the present invention may be used to detect the presence of any analyte to which the binding moiety is capable of associating (e.g. because of an affinity based binding event).
  • affinity based binding events include those between lectins and sugars, peptides and proteins, macrocyclic ligands and organic molecules.
  • the sensors of the present invention can be used to transduce these binding events in order to detect such analytes in a sample, for example, sensors in accordance with the present invention can be used to detect lectins or sugars in a sample, peptides or proteins in a sample, or macrocyclic ligands or organic molecules in a sample. Sensors in accordance with the present invention can, for example, be used to detect one of an antibody/antigen pair (e.g.
  • pollutants such as 2,4-dinitrophenol(DNP)/antiDNP, 2,3,7,8-tetrachlorodibenzofuran (TCBF)/antiTCBF, 2,3,7,8-tetrachloro-dibenzo- p -dioxin (TCDD)/AntiTC
  • the binding moiety is preferably at least part of an antigen to which the antibody is capable of binding.
  • the binding moiety is typically an antibody which is capable of binding to the antigen.
  • the binding moiety is preferably at least part of an antigen to which the antibody is capable of binding.
  • the binding moiety in the first sensor typically comprises an epitope capable of binding to the antibody.
  • the binding moiety is the epitope.
  • the epitope provides a very high degree of selectivity for the relevant antibody, and the sensor is less susceptible to interference by other species which may be present in the sample to be tested.
  • the epitope may be chemically synthesized. That is, the binding moiety may be a chemical analogue of an epitope of the antigen. Alternatively, the epitope may be isolated from the antigen.
  • the binding moiety in the first sensor is preferably a relatively small species so as not to suppress the electrochemistry of the redox active species when the binding moiety is not bound to the antibody.
  • the binding moiety is at least part of an antigen to which the antibody is capable of binding, and where the antigen is a small molecule, the antigen itself may be part of the sensor (i.e. the binding moiety is the antigen itself).
  • the binding moiety in the first sensor is situated sufficiently proximate to the redox active species so that the binding of the antibody to the binding moiety affects the electrochemistry of the redox active species.
  • the binding moiety may, for example, be bonded directly to the redox active species.
  • the binding moiety may be bonded to the redox active species via a short length alkyl chain (e.g. C 1- 10 ) or the like.
  • the redox active species may be part of the binding moiety itself.
  • the binding moiety is an antibody which is capable of binding to the antigen.
  • the second sensor further comprises at least part of an antigen to which the antibody is capable of binding (an "antigen analogue"), and the antibody is releasably attached to the sensor because it is releasably bound to the antigen analogue.
  • the antigen analogue in the second sensor may be the same as the examples of the "binding moiety" discussed above in relation to the first sensor.
  • the antigen analogue is typically a relatively small species so that the electrochemistry of the redox active species is less suppressed when the antibody detaches from the sensor than when the antibody is bound to the antigen analogue.
  • the antigen analogue may, for example, be bonded directly to the redox active species.
  • the antigen analogue may be bonded to the redox active species via a short length alkyl chain or the like.
  • SAMs self assembled monolayers
  • a process for forming on a GC electrode a self assembled monolayer comprising a redox active species and a binding moiety will now be described to illustrate how a sensor in accordance with a preferred embodiment of the present invention can be prepared.
  • Monolayers may be formed on the surface of other types of electrode using techniques well known in the art.
  • a species that is a conduit for electron movement can be bound to the surface of a GC electrode by the electrochemical reduction at the electrode of an aryl diazonium salt that is substituted with the species.
  • a negative potential at the electrode causes the diazonium salt to reduce (with the release of N 2 ), which produces a radical on the aryl ring of the diazonium salt. Radical attack on the GC electrode surface results in the formation of a C-C bond between the electrode and the aryl ring to give a stable SAM.
  • the species When the species is bound to the 4-position of the aryl group, the species projects outwards in an approximately normal conformation from the surface of the electrode in the resultant monolayer.
  • the reaction scheme for the electrochemical reduction of a 4-substituted aryl diazonium salt is depicted in Figure 2 .
  • the substituent R is the substituent on the aryl diazonium salt, and may be a species that is a conduit for electron movement.
  • a GC electrode may be modified with an aryl diazonium salt having a functional group capable of undergoing subsequent reactions at the 4-position of the aryl group.
  • a species that is a conduit for electron movement may then be bonded to the 4-position of the aryl group (and hence the electrode) in a reaction subsequent to the reaction in which the aryl diazonium salt is reduced onto the surface of the electrode.
  • the SAM formed on the surface of the GC electrode includes a mixture of species that are a conduit for electron movement and one or more kinds of blocking agents (sometimes referred to below as “insulators").
  • the blocking agents effectively "insulate” the remainder of the surface of the electrode by preventing any species which may be present in the sample to be tested from adsorbing on to the electrode. The risk of such interactions interfering with the detection of the analyte (e.g. the antibody or antigen) in the sample is therefore lessened, which gives a more reliable sensor.
  • the non-specific adsorption of molecules on the surface of the electrode may be minimized by masking the surface of the electrode with blocking agents such as bovine serum albumin or a hydrophilic layer formed by a compound such as polyethylene glycol (PEG).
  • PEG comprises short chain ethylene oxide polymers that may be bound to the 4-position of some of the aryl groups attached to the surface of the GC electrode.
  • the proportion of species that are a conduit for electron movement to insulator on the SAM may, for example, be about 1:1, 1:10, 1:20, 1:50, 1:100 or 1:1000.
  • the distal end of the species that is a conduit for electron movement may be provided with a functional group (e.g. a carboxylic acid functional group) that is capable of reacting with another compound.
  • a functional group e.g. a carboxylic acid functional group
  • a compound comprising a redox active species and possessing an appropriate functional group e.g. an amine functional group in the case of ferrocenedimethylamine or flavin adenine dinucleotide
  • an appropriate functional group e.g. an amine functional group in the case of ferrocenedimethylamine or flavin adenine dinucleotide
  • Other functional groups could, of course, be utilized to enable the species that is a conduit for electron movement and a compound containing the redox active species to be joined using standard chemical techniques.
  • the binding moiety may then be bound to the redox active species.
  • one of the amine groups of ferrocenedimethylamine may be used to couple the ferrocene moiety to the distal end of the species that is a conduit for electron movement, and the other amine group may be used to couple the binding moiety to the ferrocene moiety.
  • the ferrocene moiety joins and bridges the species that is a conduit for electron movement and the binding moiety.
  • the binding moiety may alternatively be bonded to the redox active species via a short length alkyl chain, or the like.
  • another species may be bound to the redox active species, and the binding moiety then bound to that species.
  • an antigen analogue may be bonded directly to the redox active species, or bonded to the redox active species via a short length alkyl chain, or the like, and the binding moiety (an antibody) may then be bound to the antigen analogue by exposing the antigen analogue to the antibody under conditions that allow the antibody to bind to the antigen analogue.
  • the amperometric sensors of the present invention will of course typically include additional components that enable the results of the sample analysis to be viewed by the operator.
  • the sensor would typically include a source of electricity (such as a battery), a potentiostat, a signal processor and a display.
  • the electrode having the SAM described above would typically be provided as part of a test strip comprising the electrode having the SAM described above, a reference electrode and an auxiliary electrode.
  • a schematic representation of such a sensor is depicted in Figure 3 .
  • the test strip would be exposed to the test sample (e.g. a body of water, a patient's blood, a foodstuff, a drink, or an industrial or household waste sample).
  • an electrode of the present invention could be incorporated into existing sensors for detecting the presence of an analyte in a sample to provide a sensor of the present invention.
  • Electrodes A and B Schematic representations of the surfaces of the electrode of two specific embodiments of the first and second sensors are depicted in Figure 4 (labelled “Sensor A” and “Sensor B” respectively).
  • the electrodes are GC electrodes and the redox active species is a ferrocene moiety.
  • the GC electrodes have a SAM consisting of a molecular wire in the form of oligo(phenylethylene) and an "insulator” in the form of PEG chains, both of which are bonded to the 4-position of an aryl group bonded to the electrode.
  • the redox probe is a ferrocene moiety and is bonded to and bridges the distal end of the molecular wire and the binding moiety which is biotin (an antigen).
  • Sensor B includes the same electrode, SAM and redox probe, and also includes at least part of an antigen in the form of biotin.
  • the biotin in Sensor B is bound to the ferrocene moiety.
  • the binding moiety is antibiotin, which is bound to the biotin.
  • Sensors A and B are essentially the same, however, the antibiotin is bound to the biotin in Sensor B.
  • Sensor A can readily be converted into Sensor B simply by exposing Sensor A to a sample containing antibiotin.
  • Sensor B can be converted into Sensor A by exposing Sensor B to a sample containing biotin.
  • the molecular wire having the ferrocene moiety bonded to one end and the electrode bonded to its other end, enables the transduction of the biorecognition event to be transferred to the electrode.
  • the molecular wire transfers electrons rapidly and efficiently between the ferrocene moiety and the electrode.
  • Sensor A can be used to detect the presence of antibiotin in a sample by taking electrochemical measurements which indicate whether there is a change in the electrochemistry of the ferrocene moiety as a result of the antibiotin binding to the biotin, as will now be described
  • the electrode of Sensor A is immersed for 20 mins in a test sample. During this time, if there is any antibiotin in the sample, at least some of the antibiotin will bond to the biotin on the sensor, resulting in the electrochemistry of the ferrocene moiety being suppressed. This decrease in electrochemistry is detectable and can be quantified using electrochemical techniques well known in the art.
  • Sensor B can be used to detect the presence of the antigen (i.e. biotin) in a sample by taking electrochemical measurements which indicate whether there is a change in the electrochemistry of the ferrocene moiety as a result of the antibiotin disassociating from the biotin bound to the sensor, as will now be described.
  • the antigen i.e. biotin
  • the electrode of Sensor B is immersed for 20 mins in a test sample. During this time, if there is any biotin in the sample, at least some of the antibiotin bonded to the biotin in the sensor will detach from the biotin in the sensor and bond to the free biotin in the sample.
  • the electrochemistry of the ferrocene moiety will increase because it is no longer engulfed by the antibiotin and ions may now interact with it. This increase in electrochemistry is detectable and can be quantified using electrochemical techniques well known in the art.
  • the association of the analyte with the binding moiety affects the electrochemistry of the redox active species, and thus causes an alteration in the ability of the electrode to oxidise and reduce the redox active species. For example, upon sweeping the potential of the electrode of a sensor of the present invention progressively more positive (cyclic voltammetry), or stepping the potential progressively more positive (Osteryoung square wave voltammetry), the redox active species becomes more susceptible to oxidation and eventually oxidises (e.g.
  • a ferrocene moiety will be oxidised to the ferrocinium ion). In the voltammograms, this is represented by an increase in anodic current as electrons transfer to the electrode. Sweeping the potential back more negatively will result in the reduction of the redox active species (e.g. the reduction of the ferrocinium ion to the ferrocene moiety) as electrons transfer from the electrode to the redox active species.
  • the association of the binding moiety with the analyte (for example, in the first sensor, the binding of the antibody to the binding moiety) will affect the ability of the electron transfer to occur, increasing or diminishing the peaks observed in the voltammograms.
  • the electrochemical sensors of the present invention it is also possible to quantify the amount of analyte present in a sample using the electrochemical sensors of the present invention.
  • interaction between the first sensor and the complementary antibody results in the antibody binding to the binding moiety.
  • the formed bulky structure of the binding moiety/antibody biomolecular pair perturbs the electrical communication between the redox active species and the sample, and the resulting amperometric signal is inhibited.
  • the extent of the electrode coverage by the antibody is proportional to the antibody concentration in the sample and to the time for which the electrode is exposed to the sample. Thus, if the duration of exposure to the sample is fixed, the decrease in the electrode amperometric response correlates with the antibody concentration in the sample.
  • Tetrabutylammonium tetrafluoroborate (NBu 4 BF 4 ), sodium tetrafluoroborate (NaBF 4 ), para-aminobenzoic acid, aniline, 4-mercaptobenzoic acid, 1-propanethiol, ferricyanide (K 4 Fe(CN) 6 ), 1,3-Dicyclohexylcarbodiimide (DCC), ferrocenecarboxaldehyde, sodium cyanoborohydride, acetonitrile (CH 3 CN, HPLC grade), and absolute ethanol were obtained from Sigma Chem. Co. (Sydney, Australia). Sulfo-NHS-Biotin was purchased from Pierce Chemical Company.
  • Anti-biotin from goat, bovine serum albumin fraction V (BSA), anti-pig IgG and free biotin were purchased from Sigma.
  • Reagent grade dipotassium orthophosphate, potassium dihydrogen orthophosphate, potassium chloride, sodium hydroxide, sodium chloride, sodium nitrite, ammonium acetate, sulphuric acid, hydrochloric acid, methanol and diethyl ether were purchased from Ajax Chemicals Pty. Ltd. (Sydney, Australia).
  • Single walled carbon nanotubes were obtained from Carbon Nanotechnologies Inc, USA. All reagents were used as received, and aqueous solutions were prepared with purified water (18 M ⁇ cm -1 , Millipore, Sydney, Australia).
  • the phosphate buffered saline (PBS) solutions described below were 0.15 M NaCl and 0.1 M K 2 HPO 4 /KH 2 PO 4 and adjusted with NaOH or HCl solution to pH 7.3.
  • the phosphate buffer solutions described below were 0.05 M KCl and 0.05 M K 2 HPO 4 /KH 2 PO 4 and adjusted with NaOH or HCl solution to pH 7.0.
  • GC electrodes were polished successively in 1.0, 0.3, and 0.05 micron alumina slurries made from dry Buehler alumina and Milli-Q water on microcloth pads (Buehler, Lake Bluff, IL, USA). The electrodes were thoroughly rinsed with Milli-Q water and sonicated in Milli-Q water for 5 min between polishing steps. Before derivatization, the electrodes were dried in an argon gas stream.
  • the diazonium salt solution contained a mixture of diazonium molecules substituted at the 4-position by molecular wire molecules (MW) and PEG molecules in a molar ratio of 1: 20.
  • the MW used were prepared from methyl 2'-nitro-4,4'-diphenylethynyl-4"-benzoic acid benzenediazonium tetrafluoroborate using the methods disclosed by Tour and co-workers ( Chem. Eur. J. 2001, 7, 5118-5134 ; Organic Letters 2001, 3, 993-995 ).
  • the diazonium salt solution was deaerated with argon for at least 15 min prior to derivatization.
  • the surfaces of the resultant electrodes have a SAM comprising the MW molecules and the "insulating" PEG molecules bound to the surface via the aryl group.
  • the proportion of MW:PEG molecules on the SAM was about 1:20.
  • Electrodes were rinsed with copious amounts of acetonitrile and then water, and dried under a stream of argon prior to the next step.
  • Covalent attachment of ferrocenedimethylamine to the carboxylic acid terminated molecular wire molecules on the surface of the modified electrode was achieved by dipping the electrode into an absolute ethanol solution containing of 40 mM of 1,3-dicyclohexylcarbodiimide (DCC) and 5 mM ferrocenedimethylamine overnight.
  • DCC 1,3-dicyclohexylcarbodiimide
  • the modified GC electrode of the previous step was immersed in a 1 mg mL -1 solution of NHS-biotin (NHS-biotin is a biotin molecule modified so it has a functional group that is susceptible to nucleophilic attack from amine groups) in PBS (pH 7.3) for 2 h at 4°C in order to bond the NHS-biotin to the free terminal amines of the surface bound ferrocene moieties.
  • the resultant electrode may be used in an embodiment of the sensor A described above.
  • the electrode was then rinsed with copious amounts of water and PBS, and immersed into a solution containing 0.5 ⁇ M anti-biotin (the antibody of biotin) dissolved in PBS for 20 min at 4°C.
  • the antibiotin bound to and remained bonded to the biotin labelled GC electrode because of the strong affinity between the biotin/antibiotin biomolecular pair.
  • the resultant electrode may be used in an embodiment of the sensor B described above.
  • Example 2 detecting the attachment and detachment of antibiotin
  • Figure 5 shows the cyclic voltammograms (CVs) of the modified GC electrodes described in Example 1 after the step-wise attachment of (i) ferrocenedimethylamine, (ii) biotin, and (iii) antibiotin in 0.05 M phosphate buffer (0.05 M KCl, pH 7.0) at a scan rate of 100 mV s -1 ;
  • the CV of the modified GC electrode did not change significantly.
  • the CV of the GC electrode demonstrated a pronounced reduction in peak currents.
  • OSW voltammograms (which are a more sensitive electrochemical technique) of the same modified GC electrodes also demonstrated a pronounced reduction in peak currents after incubation with anti-biotin.
  • Example 3 quantification of the concentration of anti-biotin in a sample
  • Example 1 To determine the sensitivity of the sensor A of Example 1 for detecting an antibody, the sensor was tested using samples containing different concentrations of antibiotin.
  • FIG. 7 shows the calibration curve for the detection of antibiotin using two similar electrodes (labelled "Electrode 1" and "Electrode 2") prepared as described in Example 1. The lowest detected concentration of antibiotin was 5 ng mL -1 (i.e. ⁇ 5 ppb)
  • Example 1 The reversibility of the sensor B of Example 1 was demonstrated by adding free biotin to a sample in which sensor B was immersed.
  • the relative current (obtained by dividing the current before the sensor was exposed to the sample by the current after exposure) versus time is shown in Figure 8 .
  • an increase in current density was observed upon the addition of free biotin, which is consistent with the displacement of antibiotin from the sensor.
  • the same sensor was left in the phosphate buffer solution but free biotin was not added.
  • no increase in current density was observed with time, which is consistent with the antibiotin remaining bound to the biotin and attached to the sensor.
  • Example 5 quantification of the concentration of biotin in a sample
  • Example 1 To determine the sensitivity of the sensor B of Example 1 for detecting an antigen, the sensor was tested using samples containing different concentrations of biotin. Immersion of the electrode into a buffer solution with stepwise addition of biotin between ⁇ 30 ng mL -1 to 0.3 mg mL -1 resulted in a stepwise increase in the OSWV peak current of the ferrocene molecule.
  • Figure 9 shows the calibration curve for the detection of biotin using two similar electrodes (labeled "Electrode 1" and "Electrode 2") prepared as described in Example 1. The lowest detected concentration of biotin was 30 ng mL -1 (i.e. 30 ppb)
  • Example 6 using a carbon nanotube as the conduit for electron transfer
  • SWNTs single walled carbon nanotubes
  • An initial step requires the carbon nanotubes to be shortened into manageable lengths.
  • the shortening process was performed by weighing 10 mg of pristine SWNT into a 250 mL flat bottom flask containing 40 mL of a 3:1 v/v solution of concentrated sulfuric acid (98%) and concentrated nitric acid (70%), and sonicated in a water bath at 35-40°C for 4 hours.
  • the resultant suspension was then diluted to 200 mL with water.
  • the cut SWNTs were collected on a 220 nm pore filter membrane (type: Millipore) and washed with 10 mM NaOH aqueous solution, followed by Milli-Q water until the pH of the filtrate reached 7.0.
  • the cut SWNTs were dispersed in DMF and formed a very good suspension without any aid of surfactant.
  • the cutting process results in carboxylic acid moieties being present on the ends of the carbon nanotubes.
  • the modification of glassy carbon electrodes with aligned SWNTs first requires the glassy carbon electrode to be modified with an amine terminated SAM.
  • This amine terminated SAM was produced via the reductive adsorption of 4-aminophenyl diazonium salt onto the electrode surface as described in Example 1.
  • the SWNTs are covalently attached to the 4-aminophenyl modified glassy carbon electrode by dispersing the cut SWNT (0.2 mg) in 1 mL of dimethylformamide (DMF) with 0.5 mg of dicyclohexyl carbodiimide (DCC). DCC converts the carboxylic acid group at the end of the shortened SWNTs into an active carbodiimide ester.
  • DMF dimethylformamide
  • DCC dicyclohexyl carbodiimide
  • the 4-aminophenyl modified GC electrode was then placed in the SWNT solution for 6 hours, during which the amine at the terminus of the monolayer on GC surfaces forms an amide bond with one end of each nanotube.
  • Ferrocene dimethylamine and NHS-biotin were subsequently attached to the ends of the SWNTs in the same manner to that described in Example 1.
  • Figure 10 shows the OSWVs of the modified GC electrodes described above after the step-wise attachment of (i) ferrocenedimethylamine, (ii) biotin, and (iii) antibiotin in 0.05 M phosphate buffer (0.05 M KCl, pH 7.0).
  • 0.05 M phosphate buffer 0.05 M KCl, pH 7.0
  • the sensitivity of the sensor described above i.e. in which the conduit for electron transfer is a SWNT
  • the sensor was tested using samples containing different concentrations of antibiotin.
  • Immersion of the electrode into a buffer solution with stepwise addition of antibiotin between ⁇ 10 ng mL -1 to 500 ng mL -1 resulted in a stepwise decrease in the OSWV peak current of the ferrocene molecule.
  • Figure 11 shows the calibration curve for the detection of antibiotin using two similar electrodes (labeled "Electrode 1" and "Electrode 2") prepared as described above. The lowest detected concentration of antibiotin was 10 ng mL -1 (i.e. 10 ppb).

Claims (15)

  1. Capteur électrochimique ampérométrique pour détecter un analyte, le capteur comprenant :
    - une électrode ;
    - une espèce à activité redox qui est liée à l'électrode par une espèce qui est un conducteur pour le mouvement d'électrons et est accessible de manière électrochimique à l'électrode ; et
    - une fraction de liaison capable de s'associer à l'analyte, la fraction de liaison étant suffisamment proche de l'espèce à activité redox de sorte que l'association de la fraction de liaison à l'analyte altère l'électrochimie de l'espèce à activité redox ;
    dans lequel un changement de l'électrochimie de l'espèce à activité redox a lieu quand la fraction de liaison s'associe à l'analyte.
  2. Capteur électrochimique selon la revendication 1 pour détecter un anticorps, le capteur comprenant :
    - une électrode ;
    - une espèce à activité redox qui est accessible de manière électrochimique à l'électrode ; et
    - une fraction de liaison capable de se lier à l'anticorps ;
    dans lequel la liaison de l'anticorps à la fraction de liaison altère l'électrochimie de l'espèce à activité redox.
  3. Capteur selon la revendication 2, dans lequel la fraction de liaison est au moins une partie d'un antigène.
  4. Capteur selon la revendication 3, dans lequel la fraction de liaison comprend un épitope capable de se lier à l'anticorps.
  5. Capteur selon la revendication 4, dans lequel la fraction de liaison consiste en l'épitope.
  6. Capteur selon la revendication 3, dans lequel la fraction de liaison consiste en un antigène auquel se lie l'anticorps.
  7. Capteur selon l'une quelconque des revendications 2 à 6, dans lequel la fraction de liaison est liée directement à l'espèce à activité redox ou liée à l'espèce à activité redox par une chaîne alkyle en C1 à 10.
  8. Capteur électrochimique selon la revendication 1 pour détecter un antigène, le capteur comprenant :
    - une électrode ;
    - une espèce à activité redox qui est accessible de manière électrochimique à l'électrode ; et
    - une fraction de liaison qui est un anticorps capable de se lier à l'antigène ;
    dans lequel la séparation de l'anticorps et du capteur altère l'électrochimie de l'espèce à activité redox.
  9. Capteur selon la revendication 8, qui comprend en outre au moins une partie d'un antigène, dans lequel ladite au moins une partie d'un antigène est liée à l'espèce à activité redox et la fraction de liaison est liée de manière détachable à ladite au moins une partie d'un antigène.
  10. Capteur selon la revendication 9, dans lequel ladite au moins une partie d'un antigène comprend un épitope de l'antigène.
  11. Capteur selon la revendication 9 ou la revendication 10, dans lequel ladite au moins une partie d'un antigène est liée directement à l'espèce à activité redox ou liée à l'espèce à activité redox par une chaîne alkyle en C1 à 10.
  12. Procédé pour détecter la présence d'un analyte dans un échantillon, le procédé comprenant les étapes consistant à :
    - exposer à l'échantillon une fraction de liaison capable de s'associer à l'analyte, la fraction de liaison étant associée à une espèce à activité redox qui est liée à une électrode par une espèce qui est un conducteur pour le mouvement d'électrons et est accessible de manière électrochimique à l'électrode, la fraction de liaison étant suffisamment proche de l'espèce à activité redox de sorte que l'association de la fraction de liaison à l'analyte altère l'électrochimie de l'espèce à activité redox, dans lequel un changement de l'électrochimie de l'espèce à activité redox a lieu lorsque la fraction de liaison s'associe à l'analyte ; et
    - prendre des mesures électrochimiques ampérométriques qui indiquent s'il y a un changement de l'électrochimie de l'espèce à activité redox conséquemment à l'association de la fraction de liaison à l'analyte.
  13. Procédé selon la revendication 12 pour détecter la présence d'un anticorps dans un échantillon, le procédé comprenant les étapes consistant à :
    - exposer à l'échantillon une fraction de liaison capable de se lier à l'anticorps, la fraction de liaison étant associée à une espèce à activité redox qui est accessible de manière électrochimique à l'électrode ; et
    - prendre des mesures électrochimiques qui indiquent s'il y a un changement de l'électrochimie de l'espèce à activité redox conséquemment à la liaison de l'anticorps à la fraction de liaison.
  14. Procédé selon la revendication 12 pour détecter la présence d'un anticorps dans un échantillon, le procédé comprenant les étapes consistant à :
    - exposer à l'échantillon une fraction de liaison qui est un anticorps capable de se lier à l'antigène, la fraction de liaison étant associée à une espèce à activité redox qui est accessible de manière électrochimique à l'électrode ; et
    - prendre des mesures électrochimiques qui indiquent s'il y a un changement de l'électrochimie de l'espèce à activité redox conséquemment à la dissociation de la fraction de liaison et de l'espèce à activité redox.
  15. Electrode, dans laquelle une espèce active redox est liée à la surface de l'électrode par une espèce qui est un conducteur pour le mouvement d'électrons et une fraction de liaison capable de s'associer à un analyte est associée à l'espèce à activité redox, la fraction de liaison étant suffisamment proche de l'espèce à activité redox de sorte que l'association de la fraction de liaison à l'analyte altère l'électrochimie de l'espèce à activité redox et dans laquelle l'association de la fraction de liaison à l'analyte altère l'électrochimie de l'espèce à activité redox et est détectable par des mesures électrochimiques ampérométriques, dans laquelle un changement de l'électrochimie de l'espèce à activité redox a lieu quand la fraction de liaison s'associe à l'analyte.
EP07718585.8A 2006-03-17 2007-03-16 Capteur electrochimique Not-in-force EP2005161B1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
AU2006901394A AU2006901394A0 (en) 2006-03-17 Electrochemical sensor
PCT/AU2007/000337 WO2007106936A1 (fr) 2006-03-17 2007-03-16 Capteur electrochimique

Publications (3)

Publication Number Publication Date
EP2005161A1 EP2005161A1 (fr) 2008-12-24
EP2005161A4 EP2005161A4 (fr) 2009-05-20
EP2005161B1 true EP2005161B1 (fr) 2016-02-24

Family

ID=38521942

Family Applications (1)

Application Number Title Priority Date Filing Date
EP07718585.8A Not-in-force EP2005161B1 (fr) 2006-03-17 2007-03-16 Capteur electrochimique

Country Status (4)

Country Link
US (1) US8110079B2 (fr)
EP (1) EP2005161B1 (fr)
AU (1) AU2007229320B2 (fr)
WO (1) WO2007106936A1 (fr)

Families Citing this family (22)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8197650B2 (en) 2007-06-07 2012-06-12 Sensor Innovations, Inc. Silicon electrochemical sensors
GB2463280B (en) * 2008-09-08 2011-02-02 Schlumberger Holdings Electro-chemical sensor
TWI502195B (zh) * 2009-03-10 2015-10-01 Senova Systems Inc 在用於測量樣品中之分析物的電化學感測裝置中所用的多相分析物非敏感性電極、包含此電極的電化學感測裝置，與使用此感測裝置測量樣品中之分析物的方法
FR2943460B1 (fr) * 2009-03-18 2012-03-23 Commissariat Energie Atomique Memoire hybride moleculaire a retention de charge elevee
CN102460139B (zh) * 2009-06-08 2014-04-30 西安大略大学 鉴别靶存在情况的电化学方法和装置
US9125625B2 (en) 2010-06-10 2015-09-08 The Regents Of The University Of California Textile-based printable electrodes for electrochemical sensing
FR2962445B1 (fr) * 2010-07-08 2013-06-28 Biomerieux Sa Procede de detection et d'identification directe d'un microorganisme dans un echantillon biologique dilue dans un bouillon d'enrichissement
US9417204B2 (en) 2010-07-26 2016-08-16 Senova Systems, Inc. Analyte sensor
WO2012083258A2 (fr) * 2010-12-16 2012-06-21 Sensor Innovations, Inc. Capteurs électrochimiques
US9302908B2 (en) * 2010-12-17 2016-04-05 Georgetown University Systems and process for forming carbon nanotube sensors
WO2013029115A1 (fr) * 2011-09-01 2013-03-07 Newsouth Innovations Pty Limited Capteur de compétition électrochimique
AU2012304199B2 (en) * 2011-09-01 2015-10-01 Newsouth Innovations Pty Limited Electrochemical affinity sensor
US9737247B2 (en) 2011-09-02 2017-08-22 The Regents Of The University Of California Microneedle arrays for biosensing and drug delivery
GB2497972B (en) * 2011-12-23 2016-03-16 Schlumberger Holdings Electrochemical sensors
FR2985519B1 (fr) * 2012-01-10 2016-02-19 Biomerieux Sa Procede de detection et d'identification directe par voie optique d'un microorganisme dans un echantillon biologique
US10082501B2 (en) 2013-03-24 2018-09-25 Biomedomics, Inc. Nanotube based lateral flow device for biomarker detection
US8895340B1 (en) 2013-09-10 2014-11-25 Georgetown University Biosensor and system and process for forming
CN107500264A (zh) * 2017-08-12 2017-12-22 海南师范大学 一种榕树叶基生物质多孔碳的制备方法及其在蛋白质（酶）传感器中应用
KR102334358B1 (ko) * 2019-10-31 2021-12-02 한국생명공학연구원 Trpv1 수용체 및 링커를 포함하는 그래핀 필름, 이를 포함하는 그래핀 트랜지스터 및 바이오 센서
GB2622909A (en) 2020-07-29 2024-04-03 Biolinq Incorporated Continuous analyte monitoring system with microneedle array
CN112179971B (zh) * 2020-09-29 2022-10-11 安徽中医药大学 一种检测环维黄杨星d的电化学传感器、装置及方法
SE545874C2 (en) 2021-05-08 2024-02-27 Biolinq Incorporated Fault detection for microneedle array based continuous analyte monitoring device

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5198367A (en) * 1989-06-09 1993-03-30 Masuo Aizawa Homogeneous amperometric immunoassay
US5534132A (en) 1995-05-04 1996-07-09 Vreeke; Mark Electrode and method for the detection of an affinity reaction
US6281006B1 (en) 1998-08-24 2001-08-28 Therasense, Inc. Electrochemical affinity assay
RU2161653C2 (ru) * 1998-08-24 2001-01-10 ФАРМАКОВСКИЙ Дмитрий Александрович Способ количественного электрохимического анализа биомолекул
EP1992635B1 (fr) * 2002-12-23 2012-02-08 Dynavax Technologies Corporation Oligonucléotides à séquence immunostimulatrice et ses procédés d'utilisation

Also Published As

Publication number Publication date
EP2005161A1 (fr) 2008-12-24
US20090218239A1 (en) 2009-09-03
US8110079B2 (en) 2012-02-07
AU2007229320A1 (en) 2007-09-27
EP2005161A4 (fr) 2009-05-20
WO2007106936A1 (fr) 2007-09-27
AU2007229320B2 (en) 2013-01-10

Similar Documents

Publication Publication Date Title
EP2005161B1 (fr) Capteur electrochimique
Li et al. An electrochemical immunosensor for carcinoembryonic antigen enhanced by self-assembled nanogold coatings on magnetic particles
Jia et al. Triple signal amplification using gold nanoparticles, bienzyme and platinum nanoparticles functionalized graphene as enhancers for simultaneous multiple electrochemical immunoassay
Kavosi et al. A highly sensitive prostate-specific antigen immunosensor based on gold nanoparticles/PAMAM dendrimer loaded on MWCNTS/chitosan/ionic liquid nanocomposite
Yánez-Sedeno et al. Electrochemical sensing based on carbon nanotubes
Hasanzadeh et al. Electrochemical nanobiosensing in whole blood: Recent advances
Chen et al. Electrochemical immunosensor for carcinoembryonic antigen based on nanosilver-coated magnetic beads and gold-graphene nanolabels
Samanman et al. Highly-sensitive label-free electrochemical carcinoembryonic antigen immunosensor based on a novel Au nanoparticles–graphene–chitosan nanocomposite cryogel electrode
Santhosh et al. Human serum albumin-stabilized gold nanoclusters act as an electron transfer bridge supporting specific electrocatalysis of bilirubin useful for biosensing applications
US7045364B2 (en) Electrochemical immunoassays using colloidal metal markers
Liu et al. Fabrication of an ultrasensitive electrochemical immunosensor for CEA based on conducting long-chain polythiols
US20140174950A1 (en) Electrochemical competition sensor
Wang et al. Ultrasensitive detection of carcinoembryonic antigen by a simple label-free immunosensor
Salimi et al. Highly sensitive electrochemical aptasensor for immunoglobulin E detection based on sandwich assay using enzyme-linked aptamer
Li et al. A new electrochemical immunosensor for sensitive detection of prion based on Prussian blue analogue
Song et al. Multilayer structured amperometric immunosensor based on gold nanoparticles and Prussian blue nanoparticles/nanocomposite functionalized interface
Li et al. A sensitive label-free amperometric CEA immunosensor based on graphene-Nafion nanocomposite film as an enhanced sensing platform
Li et al. Sensitive electrochemical immunosensor for cancer biomarker with signal enhancement based on nitrodopamine-functionalized iron oxide nanoparticles
Yuan et al. A simultaneous electrochemical multianalyte immunoassay of high sensitivity C-reactive protein and soluble CD40 ligand based on reduced graphene oxide-tetraethylene pentamine that directly adsorb metal ions as labels
Song et al. Simultaneous immobilization of glucose oxidase on the surface and cavity of hollow gold nanospheres as labels for highly sensitive electrochemical immunoassay of tumor marker
Zhang et al. Highly sensitive electrochemical detection of immunospecies based on combination of Fc label and PPD film/gold nanoparticle amplification
Naderi et al. Poly-L-serine/AuNPs/MWCNTs as a platform for sensitive voltammetric determination of progesterone
Nxele et al. The effects of the composition and structure of quantum dots combined with cobalt phthalocyanine and an aptamer on the electrochemical detection of prostate specific antigen
Wang et al. Sensitive electrochemical immunoassay for 2, 4, 6-trinitrotoluene based on functionalized silica nanoparticle labels
Habibi et al. A novel label-free electrochemical immunesensor for ultrasensitive detection of LT toxin using prussian blue@ gold nanoparticles composite as a signal amplification

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20081015

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LI LT LU LV MC MT NL PL PT RO SE SI SK TR

RIC1 Information provided on ipc code assigned before grant

Ipc: G01N 33/542 20060101ALI20090409BHEP

Ipc: G01N 33/543 20060101ALI20090409BHEP

Ipc: G01N 33/50 20060101AFI20071011BHEP

Ipc: G01N 27/30 20060101ALI20090409BHEP

A4 Supplementary search report drawn up and despatched

Effective date: 20090421

17Q First examination report despatched

Effective date: 20090803

DAX Request for extension of the european patent (deleted)
REG Reference to a national code

Ref country code: DE

Ref legal event code: R079

Ref document number: 602007044956

Country of ref document: DE

Free format text: PREVIOUS MAIN CLASS: G01N0033500000

Ipc: G01N0033542000

RIC1 Information provided on ipc code assigned before grant

Ipc: G01N 33/542 20060101AFI20150527BHEP

Ipc: G01N 33/543 20060101ALI20150527BHEP

Ipc: G01N 33/68 20060101ALI20150527BHEP

GRAP Despatch of communication of intention to grant a patent

Free format text: ORIGINAL CODE: EPIDOSNIGR1

INTG Intention to grant announced

Effective date: 20150716

GRAS Grant fee paid

Free format text: ORIGINAL CODE: EPIDOSNIGR3

INTG Intention to grant announced

Effective date: 20151216

GRAA (expected) grant

Free format text: ORIGINAL CODE: 0009210

AK Designated contracting states

Kind code of ref document: B1

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LI LT LU LV MC MT NL PL PT RO SE SI SK TR

REG Reference to a national code

Ref country code: GB

Ref legal event code: FG4D

REG Reference to a national code

Ref country code: CH

Ref legal event code: EP

REG Reference to a national code

Ref country code: AT

Ref legal event code: REF

Ref document number: 776992

Country of ref document: AT

Kind code of ref document: T

Effective date: 20160315

REG Reference to a national code

Ref country code: IE

Ref legal event code: FG4D

REG Reference to a national code

Ref country code: FR

Ref legal event code: PLFP

Year of fee payment: 10

REG Reference to a national code

Ref country code: DE

Ref legal event code: R096

Ref document number: 602007044956

Country of ref document: DE

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: GB

Payment date: 20160310

Year of fee payment: 10

REG Reference to a national code

Ref country code: LT

Ref legal event code: MG4D

REG Reference to a national code

Ref country code: NL

Ref legal event code: MP

Effective date: 20160224

REG Reference to a national code

Ref country code: AT

Ref legal event code: MK05

Ref document number: 776992

Country of ref document: AT

Kind code of ref document: T

Effective date: 20160224

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: ES

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160224

Ref country code: FI

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160224

Ref country code: GR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160525

Ref country code: IT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160224

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: DE

Payment date: 20160329

Year of fee payment: 10

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: LV

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160224

Ref country code: AT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160224

Ref country code: PL

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160224

Ref country code: LT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160224

Ref country code: BE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20160331

Ref country code: SE

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160224

Ref country code: NL

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160224

Ref country code: PT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160624

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: FR

Payment date: 20160325

Year of fee payment: 10

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: DK

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160224

Ref country code: EE

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160224

REG Reference to a national code

Ref country code: CH

Ref legal event code: PL

REG Reference to a national code

Ref country code: DE

Ref legal event code: R097

Ref document number: 602007044956

Country of ref document: DE

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: CZ

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160224

Ref country code: RO

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160224

Ref country code: SK

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160224

REG Reference to a national code

Ref country code: IE

Ref legal event code: MM4A

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: BE

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160224

PLBE No opposition filed within time limit

Free format text: ORIGINAL CODE: 0009261

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: NO OPPOSITION FILED WITHIN TIME LIMIT

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: LI

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20160331

Ref country code: IE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20160316

Ref country code: CH

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20160331

26N No opposition filed

Effective date: 20161125

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: BG

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160524

Ref country code: SI

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160224

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: MT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160224

REG Reference to a national code

Ref country code: DE

Ref legal event code: R119

Ref document number: 602007044956

Country of ref document: DE

GBPC Gb: european patent ceased through non-payment of renewal fee

Effective date: 20170316

REG Reference to a national code

Ref country code: FR

Ref legal event code: ST

Effective date: 20171130

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: DE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20171003

Ref country code: FR

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20170331

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: GB

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20170316

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: CY

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160224

Ref country code: HU

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT; INVALID AB INITIO

Effective date: 20070316

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: LU

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20160316

Ref country code: MC

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160224

Ref country code: TR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160224

Ref country code: IS

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160224

Ref country code: MT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160331